The attachment of dissimilar materials is a major engineering challenge because of the high levels of localized stress that develop at such interfaces. An effective biologic solution to this problem can be seen at the attachment of tendon (a compliant, structural "soft tissue") to bone (a stiff, structural "hard tissue"). The unique transitional tissue that exists between uninjured tendon and bone is not recreated during tendon-to-bone healing. Surgical reattachment of these two dissimilar biologic materials therefore often fails. We propose to examine the development of the tendon-to-bone insertion site and use results to guide tissue engineering studies for tendon-to-bone repair. The tendon-to-bone insertion site is a hierarchical composite material with complex structural organization, compositional makeup, and biomechanical behavior. Most previous research has focused on analysis at the tissue (i.e., millimeter scale) and microscopic (i.e., micrometer scale) levels. Less is known about the nanoscale architecture of the insertion site constituents - hydroxyapatite (i.e., mineral), fibrocartilage, and collagen fibers. Furthermore, little is known about the transition in chemistry and structure across the tendon- to-bone interface and how these parameters affect the mechanical response of the system. Our preliminary studies indicate that there is a smooth transition in mineral and fibrocartilage concentration between tendon and bone, and that this transition may partly explain the mechanical behavior at the tissue level. In Aim 1 we will study the development of the natural tendon-to-bone interface between the rat supraspinatus tendon and humeral head. In Aim 2 we will synthesize collagen matrices in a controlled in vitro system with concentration gradations in mineral and fibrocartilage. Data gathered from these two aims will be used to test hypotheses related to the role of mineral and fibrocartilage at the tendon-to-bone insertion. The hydroxyapatite to collagen ratio will be investigated using Raman spectroscopy. The interface between collagen and hydroxyapatite will be characterized using electron microscopy. Localized gene expression will be evaluated using in situ hybridization. Data will be interpreted in the context of stress transfer using biomechanical models. PUBLIC HEALTH RELEVANCE: Musculoskeletal injuries are a common cause of pain and disability, and result in significant health care costs.1 Injuries to the soft tissues often require surgical repair and tendon-to-bone healing (e.g., rotator cuff repair2,3, anterior cruciate ligament reconstruction4,5, flexor tendon avulsion6). Clinical outcomes have frequently been disappointing (e.g., at the rotator cuff the recurrence of tears to repaired tendons has been reported to be as high as 94%3,7,8). The work in this proposal will form the foundation for future surgical reconstruction techniques that will improve healing by regenerating the natural tendon-to-bone insertion.